In Vivo Tracking of Multiple Tumor Exosomes Labeled by

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In Vivo Tracking of Multiple Tumour Exosomes Labeled by Phospholipid-Based Bioorthogonal Conjugation Pengjuan Zhang, Bo Dong, Erzao Zeng, Fengchao Wang, Ying Jiang, Dianqi Li, and Dingbin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01506 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Analytical Chemistry

In Vivo Tracking of Multiple Tumour Exosomes Labeled by Phospholipid-Based Bioorthogonal Conjugation Pengjuan Zhang,† Bo Dong,† Erzao Zeng,† Fengchao Wang,† Ying Jiang,† Dianqi Li,†and Dingbin Liu*,†,§ †

College of Chemistry, Research Center for Analytical Sciences, State Key Laboratory of Medicinal Chemical Biology, and Tianjin Key Laboratory of Molecular Recognition and Biosensing, Nankai University, Tianjin 300071 (China) § Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071 (China) Supporting Information Placeholder ABSTRACT: Exosomes are cell-secreted nanoscale membrane vesicles that play critical roles in many pathophysiological processes. The clinical value of exosomes is under intense investigation, yet current knowledge regarding their in vivo properties is very limited due to the lack of efficient labeling techniques. Here, we report a phospholipid-based bioorthogonal labeling strategy to endow exosomes with optical probes without influencing their native biological functions. We investigated the dynamic in vivo biodistribution and organotropic uptake of multiple tumour exosomes in a single mouse. The results indicate that the exosomes derived from different cell lines show specific organotropic uptake. This phospholipid-based labeling strategy opens a new window to directly visualize and monitor the exosomes trafficking in live systems and holds great promise for exploring exosome-involved biological events such as cancer metastasis.

INTRODUCTION Exosomes are 30-150 nm diameter extracellular vesicles secreted by mammalian cells and act as communicators for intercellular transmission of biological signals.1-3 They are involved in a variety of pathophysiological events such as cancer metastasis, inflammation, and tissue regeneration.4-6 For example, exosomes can transfer malignant phenotype to recipient cells and establish a pre-metastatic site to prompt cancer cell growth.7,8 Recently, exosomes have been recognized as promising non-invasive cancer biomarkers.9,10 Further, as a type of naturally generated nanovesicles, exosomes are excellent drug delivery carriers showing many advantages (e.g., high stability and biocompatibility) over its artificial synthetic counterpart.11 Understanding the in vivo properties of exosomes could not only facilitate deciphering the mystery of cancer metastasis,6 but also help to design targeted delivery systems for therapeutic purpose. However, due to the lack of efficient tracking techniques, the in vivo properties of exosomes have not been well explored yet. This has largely hampered the biological investigation and biomedical application of exosomes. Labeling exosomes with molecular imaging probes is a prerequisite for investigating their in vivo behaviors. However, because of their small size and complexity of membrane surface chemistry, it remains a great challenge to label exosomes with proper chemistry. The most widely used strategy for exosome labeling is genetic engineering with fluorescent proteins12,13 or bioluminescence reporters,14,15 which is a tedious and timeconsuming process. Moreover, the fused fluorescent proteins may affect the exosome biogenesis.16 Direct labeling of the membrane proteins on exosomes has been attempted.17,18 This strategy could impair biological function of the modified exosomes by altering or obscuring the active site of membrane proteins.16 Furthermore, the heterogeneity of protein expression on different exosomes enables the protein labeling strategy unreliable particularly when the protein expression is low.19 Another commonly used method is non-covalent incorporation of lipophilic dyes (e.g., PKH26)20,21

or carbocyanine dyes (e.g., DIR and DIO)22,23 into the lipid bilayers of exosome membranes. Such dyes are prone to be released from the exosomes in the process of in vivo trafficking, providing misleading information.24-26 Recently, nanoparticles have been loaded into exosomes as tracking reagents.27,28 The relatively large size of nanoparticles may inevitably influence the structural integrity and functionality of exosomes. Given these limitations, the development of a robust, biocompatible, and simple labeling method would open the door to a more detailed understanding of the in vivo exosome trafficking as well as their biological functions. In this study, we describe a phospholipid-based bioorthogonal chemistry for labeling exosomes (Figure 1) and investigating their in vivo biodistribution and organotropic uptake. We envision that phospholipids are ideal species for exosome labeling because they are the basic building blocks of all biological membranes.29 More importantly, in contrast to protein labeling, phospholipid labeling will not affect the intrinsic activity of exosomes.25 Unfortunately, phospholipids are inert biomolecules and have no active chemical groups for covalent conjugation. Here, we adopted a metabolic strategy to incorporate exosome phospholipids with an unnatural choline analogue bearing an azide (N3, a typical bioorthogonal functional group). Subsequently, the immobilized azide was conjugated with a fluorescent probe using a bioorthogonal reaction (e.g., copper-free click chemistry), a biocompatible conjugation strategy pioneered by Bertozzi et al.30,31 This strategy has been widely used to study the biochemistry of various cellular components in living systems.32-41 Through intravenous injection of several types of labeled exosomes into a living mouse, their in vivo biodistribution and organotropic uptake were investigated simultaneously. Impressively, we found that the exosomes derived from MDAMB-231 and HS578T cell lines can colonize the brain astrocytes. This finding provides a new opportunity for the theranostics of astrocyte-related disorders such as gliomas, Alzheimer's disease, and stroke.

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For the preparation of azide-Cho, the 1-Br-ethyl-choline (5.13 g, 0.026 mol) and sodium azide (16.9 g, 0.26 mol) was slowly added to 90:30 mL of acetone: water while stirring. The reaction mixture was allowed to react overnight. The resulting solution was evaporated under reduced pressure, and the pure azide-Cho was extracted by dichloromethane: ethanol (10:1) and dried under vacuum to yield white solid azide-Cho (2.02 g, 0.0126 mol, 48%). 1 H NMR (D2O, 400 MHz): δ 3.93 (s, 2H), δ3.85 (s, 2H), δ 3.573.54 (t, 2H, J1=2.96 Hz, J2=8.49 Hz), δ 3.49-3.47 (t, 2H, J1= 2.54 Hz, J2=7.41 Hz), 3.11 (s, 6H). ESI-MS for C6H14ON4 (calculated at 159): m/z 159.1 (M+H+). Cell Culture. Mouse breast cancer cell line 4T1, human breast cancer cell lines including MCF-7 cells, HS578T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco). Cell cultures were incubated in a 5% CO2 incubator at 37 oC. MDA-MB-231 cells were cultured in Leibovitz's L-15 Medium (L-15, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin/streptomycin (Gibco) at 37 oC, 100% atmosphere inside an incubator.

Figure 1. Phospholipid-based bioorthogonal chemistry for exosome labeling. (a) Metabolic incorporation of azides into exosome phospholipids. (b) Bioorthogonal labeling of the azide-modified exosomes with DBCO-containing dyes via copper-free click chemistry.

EXPERIMENTAL SECTION Materials and Reagents. 1,2-Dibromoethane was purchased from J&K Scientific Ltd (Beijing, China). N, N-Dimethylethanolamine was purchased from Innochem (Beijing, China). Dibenzocyclooctynes-derived Cy3, Cy5, Cy7, Fluor 488 and MB594 (DBCO-Cy3, DBCO-Cy5, DBCO-Cy7, DBCO-Fluor 488, MBTM 594 DBCO) were purchased from Click Chemistry Tools (Scottsdale, AZ). DIR Iodide was purchased from Yeasen (Shanghai, China). Anti-TSG101 mouse monoclonal antibody, Alexa Fluor® 488-linked anti-GFAP rabbit monoclonal, horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG antibody, HRP-conjugated Goat Anti-Rabbit IgG antibody and HRPconjugate Anti-β-actin mouse monoclonal antibody were purchased from Abcam (Cambridge, MA). Anti-Alix mouse monoclonal antibody was purchased from Cell Signaling Technology (Beverly, MA) Anti-CD9 rabbit polyclonal antibody and AntiCalnexin rabbit polyclonal antibody were purchased from Proteintech (Chicago, IL). MCF-7, HS578T, MDA-MB-231 and 4T1 cells were obtained from Shanghai institute of life sciences, Chinese Academy of Sciences (Shanghai, China). Dulbecco's phosphate-buffered saline (D-PBS) was purchased from Genview (TX, USA). 1H NMR spectrum was recorded by a Varian 400 MHz spectrometer. JMS-LC mate mass spectrometer was applied to record electron spray ionization mass spectra (ESI-MS). Synthesis of Azide-Cho. Azide-Cho was synthesized according to reported procedures34 with a little modification. 1,2dibromoethane (43 mL, 0.499 mol) and dimethyl-ethanolamine (7.7 mL, 0.077 mol) was added to 100 mL of dry tetrahydrofuran (THF) in a round-bottom flask. The reaction mixture was stirred overnight. Then the white precipitate was washed with cold diethyl ether for three times and dried in the thermostatic vacuum drier to give white powder 1-Br-ethyl-choline (5.13 g, 0.026 mol, 34%). 1H NMR (D2O, 400 MHz): δ 4.05 (s, 2H), δ 3.88-3.85 (t, 2H, J=6.40 Hz), δ 3.80-3.76 (t, 2H, J= 7.56 Hz), δ 3.55 (s, 2H), 3.20 (s, 6H). ESI-MS for C6H14ONBr (MW: 197.1): m/z 196.03 (M-H+), m/z 198.03 (M+H+).

Mice Information. All animal studies were performed in compliance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. Female BALB/C nude mice were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) and used at 6-8 weeks of age. MTT Assay. Cytotoxicity assessment of azide-Cho was performed by MTT assay. MCF-7 cells were incubated with azideCho at different concentrations in the culture medium for 24 h. MTT solution was added into the culture medium with a final concentration of 0.5 mg/mL and the mixture was further cultured at 37 oC for 4 h. Finally, the absorbance at 490 nm for each well was measured with a microplate reader. Characterization of Azide-Modified Cells. For the lipid profiling of azide modified cells, 5 × 106 MCF-7 cells labeled with azide-Cho and untreated controls were collected and total lipids were extracted based on reported procedures.33 Briefly, cells were resuspended in 0.5 mL PBS. 1.2 mL Methanol and 300 μL chloroform were added into the cell suspension and vortexed until the solution reached a single liquid-phase. The tube was then centrifuged at 14,000 rpm for 5 min. The supernatant was transferred to a fresh tube, chloroform (600 μL) and 0.1% (v/v) aqueous acetic acid (1.2 mL) were added, vortexed and centrifuged at 14,000 rpm for 5 min. The upper aqueous phase was discarded, and the lower organic phase was transferred to a fresh 5 mL round-bottom flask and evaporated under reduced pressure. The lipid was resuspended in 200 μL of chloroform/methanol (1/1). LC-MS analysis was performed on a Waters ACQUITY UPLC SYNAPT G2Si QTOF system with BEH HILIC columns. Isolation and Characterization of Exosomes. Exosomes were purified by sequential centrifugation as previously reported.42 To get azide-labeled exosomes, MCF-7, MDA-MB-231, HS478T and 4T1 cells were incubated with 500 μM azide-Cho for 48 h in FBS deleted culture medium. For native exosomes, MDA-MB-231 cells was incubated in FBS deleted culture medium for 48 h. The cell culture supernatant was then collected and centrifuged at 1000 g for 10 min to remove any cell contamination. The supernatant was transferred into new tube and centrifuged at 2,000 g for 30 min to remove large cell debris, followed by filtration through a 0.22 μm filter membrane. Finally, exosomes were collected by spinning at 100,000 g for 70 min. Exosomes were washed in PBS and pelleted again by ultracentrifugation (Beckman 45Ti rotor). Exosome preparations were verified by transmission electron


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Analytical Chemistry microscopy (Talos F200C, FEI). Exosome size were analyzed using the nanoparticle Analyzer (Zetasizer Nano ZS, Malvern Instruments). The final exosome pellet was resuspended in PBS and protein concentration was measured by BCA (Beyotime Biotechnology).

perature for 2 h and washed three times with TBST and then incubated with secondary antibodies at room temperature for 1 h. After washing three times with TBST for 10 min each, the membrane was incubated with BeyoECL Plus (Beyotime Biotechnology) and imaged with Azure c600 (Azure Biosystems, USA).

Fluorescence Labeling of Exosomes. 200 µg of azide-labeled exosomes derived from MCF-7, HS578T, MDA-MB-231, and 4T1 cells were reacted with 1 µM DBCO-Cy7 for 1 h, respectively. 200 µg of native exosomes isolated from MDA-MB-231 cells were labeled with DIR according to the manufacture instructions. Excess Cy7 dyes were washed away through ultrafiltration (molecular weight cutoff 30,000) for 5 times. Purified exosomes were resuspended with PBS and the protein concentrations were measured by BCA again. The fluorescence profiles were obtained with F-4600 fluorescence spectrophotometer (Hitachi, Tokyo).

RESULTS AND DISCUSSION

In Vivo Imaging of the Dye-Labeled Exosomes. MCF-7 EXO-Cy7, MDA-MB-231 EXO-Cy5 and HS578T EXO-594 were injected intravenously into BALB/C nude mice (20 µg dissolved in 100 µL PBS of exosomes/mouse). After 24 h post-injection, the mice were scanned with a Maestro EX in vivo fluorescence imaging system. When taking images, the mice were temporarily anesthetized with isoflurane and placed appropriately. Then, the mice were sacrificed and the main organs were isolated and imaged. The fluorescence signals in each organ were quantified with the photons/second/steradian (ph/s/sr), which were normalized to that of the injected dose based on the fluorescence intensity and were expressed by the percentage of the injected dose per organ (% ID/organ). Biodistribution of Exosomes. The mice were first treated with 100 µL of the dye-labeled exosomes via intravenous injection. Then, the blood samples were collected at indicated time points: 0.5, 1, 2, 4, 6, and 24 h. The whole blood was placed overnight and centrifuged at 8000 g for 20 min at 4 °C. The supernatants were diluted with PBS, and their fluorescence intensity was then measured by the F-4600 fluorescence spectrophotometer. The amount of exosomes in each sample was normalized to that of the injected dose and was expressed by the percentage of the injected dose per mL (% ID/mL).

Metabolic Incorporation of Azides into Phospholipids. In many naturally occurring bilayers, the outer leaflet is mainly composed of choline-containing phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM) (typical structure is shown in Figure 2a).43,44 To metabolically modify the phospholipids, we first synthesized a choline analogue azidoethyl-choline (azide-Cho), which bears a terminal azide group and can incorporate into all types of choline-containing phospholipids in cultured living cells. The synthesis of the biosynthetic precursor azide-Cho is shown in Figure 2b. First, the tertiary amine group in dimethylethanolamine reacts with excess 1,2-dibromoethane, producing 1Br-ethyl-choline. Then, the Br group in 1-Br-ethyl-choline is replaced by azides in the presence of sodium azide (NaN3), yielding azide-Cho. Both the intermediate and product were characterized by 1H NMR and mass spectrometry (details are shown in Supplementary Figures S1-4). The biocompatibility of the assynthesized precursors was evaluated by incubating different concentrations of azide-Cho into MCF-7 cell solutions (1×105 cells/mL) for 24 h. MTT assay was employed to evaluate the cell viability of the probes. It is impressive that negligible cell toxicity can be observed even when the concentration of azide-Cho reach 5 mM, 10 folds higher than the optimized concentration (0.5 mM) that was used for cell labeling throughout the study (Supplementary Figure S5).

Immunofluorescence Detection of Brain Tissue Sections. For histological analysis, Mice were perfused with PBS and then a mix of 2% paraformaldehyde and 2.5% glutaraldehyde through the left cardiac ventricle. Brain tissues were dissected and fixed in a mix of 2 % PFA and 20% sucrose in PBS overnight. Immunofluorescence detection was performed at Beijing Medical Discovery Leader using Alexa Fluor® 488-linked anti-GFAP rabbit monoclonal. And fluorescent images were obtained using a Leica confocal microscope and analyzed using LAS X. Western Blot Assays. Exosomes or cells were lysed with RIPA buffer containing a complete protease inhibitor tablet (Beyotime Biotechnology). Lysates were centrifuged at 14,000 g for 20 min and supernatant fractions were collected and send for western blot analysis. Samples were separated by SDS-PAGE and transferred onto a 0.45-μm nitrocellulose membrane (Millipore). Membranes were blocked with 5% skim milk (in TBST, 20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) at 37 oC for 1 h before primary antibody incubation. The antibodies against the following proteins were used for western blot analysis: Anti-Alix (1:1,000, Cell Signaling; 2171), Anti-TSG101 (1: 1,000, abcam; ab83), Anti-beta Actin (1:2000, abcam; ab173838), Anti-CD9 (1:5000, proteintech; 20597-1-AP), Anti-Calnexin (1:5000, proteintech; 10427-2-AP), Anti-rabbit IgG, HRP-linked antibody (1: 3,000, abcam; ab6721) and anti-mouse IgG, HRP-linked antibody (1: 3,000, abcam; ab6789) were used as secondary antibodies. The membranes were incubated with primary antibodies at room tem-

Figure 2. (a) A typical structure of choline-containing phospholipids. (b) Synthesis of azide-Cho and metabolic production of azidoethyl-phospholipids. (c) LC-MS analysis of total phospholipids extracted from MCF-7 cells treated with or without azide-Cho. After azide-Cho incorporation, apparent shifts of LC retention time were observed from 8.2 to 6.9 min for PC species, and from 9.7 to 8.2 min for SM species. MS indicates an expected mass shift of 55 amu of cellular lipids after incorporation with azideCho.



When fed the living MCF-7 cells with the as-synthesized azideCho precursors, they can enter cells through transporters.45 After that, the azide-Cho was phosphorylated by Cho kinase (CHK). For PC biosynthesis, the activated Cho was converted to CDPCho by CTP-Cho cytidylyl transferase (CCT) and then transferred from CDP-Cho to diacylglycerol by CDP-choline-diacylglycerol phosphocholine transferase (CHPT) in the endoplasmic reticulum. For SM, the choline head group is transferred from PC to ceramide in the golgi apparatus. The PC and SM species were incorporated into membrane phospholipids via the vesicular trafficking pathway.33 As a consequence, one methyl in native choline is replaced by an azidoethyl group in the metabolic process. To verify the metabolic products, the total phospholipids were extracted from MCF-7 cells that were treated with or without azide-Cho. The generation of azidoethyl-phospholipids was analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS). After incorporation of azideCho, significant shifts in the LC retention time of the azide-PC and azide-SM species were observed from 8.2 to 6.9 min and from 9.7 to 8.2, respectively (Figure 2c, left). Compared with the native phospholipids, the azidoethyl-phospholipids showed an expected mass shift of 55 amu for all major peaks, indicating the successful incorporation of azide-Cho in both PC and SM species from the metabolic labeling samples (Figure 2c, right). Production of Bioorthogonally Labeled Exosomes. Breast cancer is the most common malignant disease within females. In this study, we chose three different breast cancer cell lines including MCF-7 (ER+, PR+, HER2-), MDA-MB-231(ER-, PR-, HER2-, metastasis), and HS578T (ER-, PR-, HER2-, primary)

cells as the donor cells to secret specific exosomes. The detailed information regarding the three cell lines are summarized in Supplementary Table S1. Exosome biogenesis are an endocytic event at the plasma membrane starting from the generation of early endosomes. After maturation to late endosomes, the intraluminal vesicles are generated by inward budding of the endosomal membrane, further forming multi-vesicular bodies (MVBs). Subsequently, vesicles are secreted through the fusing of MVBs with the plasma membrane. Ultimately, exosomes are released from donor cells. By means of the classical ultracentrifugation method, the azide-labeled exosomes were isolated from corresponding donor cells. The three types of exosomes were quantified by using protein concentration: for each sample, the yield was 100-200 µg of exosomal proteins per 300 mL cellular supernatant. Transmission electron microscopy (TEM) images shows that the diameters of the azide-labeled exosomes are all around 70 nm, while their hydrodynamic diameters recorded by dynamic light scattering (DLS) are ~100 nm (Figure 3a and Supplementary Figure S6). We further identified the isolated exosomes with western blot analysis and compared the results with corresponding cell lysates. After incubation with CD9, TSG101, and Alix antibodies (general exosome markers), three characteristic bands appeared at 25 kDa, 45 kDa, and 95 kDa respectively for all samples (Figure 3b). However, no bands can be found for the exosome samples incubated with calnexin antibodies that were applied as a negative control. These results confirm that those obtained vesicles were typical exosomes.

Figure 3. Characterizations of the dye-labeled exosomes (EXO) derived from different cancer cell lines. (a) TEM and DLS measurements of dye-labeled exosomes isolated from MCF-7, MDA-MB-231, and HS578T cells, respectively. (b) Western blot analysis of exosomes and corresponding cell lysates using Alix, TSG101 and CD9 as general exosome markers. Calnexin was adopted as a negative control while βactin was employed as a positive control. (c) Fluorescence spectra of the three types of dye-labeled exosomes.


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Figure 4. In vivo and ex vivo tracking of breast cancer cellderived exosomes. (a) The mouse received a single intravenous injection of fluorescently labeled MCF-7, MDA-MB-231, and HS578T-derived exosomes was imaged after 24 h post injection. (b) Organs harvested from the same mouse were visualized at Cy7, Cy5, and MB 594 channels using the Maestro system. (c) Ex vivo biodistribution of exosomes were analyzed and quantified by recoding the photons/second/steradian (ph/s/sr) of each organ, which was further normalized to that of the injected dose based on fluorescence intensity and was expressed by the percentage of the injected dose per organ (% ID/organ). These results are expressed as the mean ± standard deviation (n = 3). With the azide-labeled exosomes from the three cell lines in hand, we made use of bioorthogonal chemistry to conjugate the exosomes with three kinds of dibenzocyclooctyne (DBCO)conjugated dyes including DBCO-Cy7, -Cy5, and -MB 594. This conjugation is readily accomplished by simply mixing them together without resorting to additional reagents. After labeling, the excess free dyes were removed through an ultrafiltration membrane (30 kDa). The dye-conjugated exosomes were recorded by fluorescence spectrum, with characteristic emission peaks at 793, 669, and 640 nm, indicating the successful bioorthogonal labeling of Cy7, Cy5, and MB 594 on the exosomes derived from MCF-7, MDA-MB-231, and HS578T cells, respectively (Figure 3c). Note that the fluorescently-labeled exosomes are unable to be directly visualized by confocal microscopy due to the nanoscale size of exosomes that is smaller than the diffraction limit of light. Simultaneous Visualization of Multiple Dye-Labeled Exosomes in a Single Living Mouse. The successful labeling of exosomes encouraged us to investigate their in vivo behaviors. To do this, these labeled exosomes were simultaneously administrated into the tail veins of a single BALB/C mouse. After

24 h injection, the exosome biodistribution was evaluated with Cy7, Cy5, and 594 channels by optical microscopy. Through imaging the whole body of the mouse with the channels, strong fluorescence signals were detected in the brain of mouse administered with HS578T and MDA-MB-231 cell-derived exosomes (Figure 4a). Interestingly, the same mouse treated with the exosomes isolated from MCF-7 cells had no fluorescence signals in the brain. To explore the possible effect of coadministration of different exosomes on their in vivo behaviors, the Cy7-labeled exosomes derived from MCF-7, MDA-MB-231, and HS578T cells were intravenously injected into three mice independently. The independent administration showed similiar signal distribution with that from co-administration of the mixed exososmes (Supplementary Figure S7). These results confirmed that the co-administration of multiple exosomes has negligible effect on their individual biological activity. Furthermore, to examine the exosome uptake by different distant organs, the mouse was sacrificed and the organs including brain, liver, spleen, lung, and kidney were collected for ex vivo imaging (Figure 4b and Supplementary Figure S9). We observed that MCF-7-derived exosomes were more efficiently uptaken in kidney than that in other organs. Impressively, for both MDA-MB-231 and HS578T cell-secreted exosomes, obvious uptake in the brain was observed (Figure 4c). The coadministration strategy shows the similar exosome uptake with that based on independent administration (Supplementary Figure S8), agreeing well with the in vivo imaging results. To verify the reliability of the labeling strategy, we used commercially available membrane fluorescent dye DIR to label native exosomes derived from MDA-MB-231 cells and compared with our click chemistry-based method. DIR can be incorporated into the lipid bilayers of MDA-MB-231 exosome membranes for fluorescent imaging. Both the two labeling strategies show the similar tendency in the organ-dependent uptake (Supplementary Figure S10). Moreover, our labeling strategy can be extended to study the exosomes derived from another breast cancer cell line— 4T1 cells, and the organotropic uptake is in accordance with the previous report (Supplementary Figure S11).46 Time course of blood concentrations of the Cy7-labeled exosomes were further evaluated at desired intervals after the intravenous injection. The serum fluorescent intensity of Cy7 decreased quickly for all the exosomes and only 1% injected dose/mL (% ID/mL) of exosomes can be detected in the blood after 6 h post injection (Supplementary Figure S12). We paid particular attention to the brain-tropic uptake of HS578T and MDA-MB-231 cell-derived exosomes since the early diagnosis and treatment of brain-related diseases such as gliomas, Alzheimer's disease, and stroke remain a great challenge to date. To gain insight into what kind of recipient cells that the MDAMB-231 and HS578T cell-derived exosomes colonized in the mouse brain, the brain tissues were allowed for histological analysis. Imaging results showed that exosomes isolated from these two cell lines could target to specific regions in the brain slice. Compared with region 1 (R1), the Cy5 signals (red, derived from MDA-MB-231 exosomes) were mainly detected on the starshaped cells in region 2 (R2) as shown in Figure 5a. Immunofluorescence analysis was performed by staining glia fibrillary acidic protein (GFAP), an intermediate filament expressed in astrocyte cells. The results showed that the Cy5 signals were fully overlapped with fluorescence of the GFAPpositive astrocyte cells (Figure 5b). The similar results were observed from the MB 594 signals derived from HS578T exosomes (Supplementary Figure S13), indicating that the exosomes secreted by MDA-MB-231 and HS578T cell lines were most likely uptaken by astrocytes in the mouse brain. The braintropic exosome uptake we found in this study could not only



provide an opportunity to reveal the mechanism of brain-tropism cancer metastasis but also open a new door to the theranostics of astrocyte-related disorders. Our future study will focus on the essential factors that mediate the brain-tropic uptake.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This study was supported by the National Natural Science Foundation of China (21475066 and 21775075) and the Thousand Youth Talents Plan of China.

REFERENCES (1) (2) (3) (4) (5) (6)

Figure 5. Biodistribution of MDA-MB-231 cell-derived exosomes in the mouse brain tissue slice. (a) Confocal imaging of brain tissue slice showed that the Cy5 signals derived from the MDA-MB-231 cell-derived exosomes were distributed specifically on the star-shaped cells in the region 2 (R2). (b) Co-staining of astrocyte-specific GFAP and Cy5 signals.

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CONCLUSIONS In summary, we proposed a phospholipid-based bioorthogonal conjugation strategy for labeling multiple exosomes and investigated their in vivo biodistribution and organotropic uptake in a single mouse. This strategy does not involve any reagents or procedures that may influence the activity of exosomal proteins or destroy the structural integrity of exosomes, thus retaining their intrinsic functions. By means of this efficient labeling strategy, we found that the exosomes derived from MDA-MB-231 and HS578T cell lines can target to the astrocytes in the mouse brain. We believe that this important finding may open a new opportunity for both basic and applied studies of exosomes. Our following study will combine this novel labeling strategy with proteomics to further investigate the intrinsic mechanism of braintropic uptake. Overall, our strategy not only provides an excellent means for in vivo exosome tracking, but also offers a new platform to reveal novel mechanisms, to identify potential biomarkers, and to deliver therapeutic reagents in living systems.4749

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Supporting Information

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Supplementary figures and tables are included in the Supporting Information. This material is available free of charge via the internet at http://pubs.acs.org.

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AUTHOR INFORMATION Corresponding Author *[email protected].

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